Expression of the pRb-Binding Regions of E1A Enables Efficient Transformation of Primary Epithelial Cells by v-src

Copyright © 1998, American Society for Microbiology

Expression of the pRb-Binding Regions of E1A Enables

Efficient Transformation of Primary Epithelial Cells by v-src

ROBERT S. FISCHER†

MARGARET P. QUINLAN*

Department of Microbiology and Immunology, University of Tennessee Health Science Center,

Memphis, Tennessee 38163

Received 29 September 1997/Accepted 23 December 1997

Primary cultures of rat embryo fibroblasts have been shown to be resistant to transformation by dominant

oncogenes such as v-src. We sought to determine if similar resistance is displayed by primary epithelial cells,

and, if so, whether an immortalizing oncogene such as E1A could enhance transformation of primary epithelial

cells by v-src. Transformation of primary rat epithelial cells by v-src was synergistically enhanced when E1A

expression plasmids were cotransfected with a v-src expression plasmid. Foci were more numerous and

observed earlier (9 to 14 days) with E1A plus v-src than with v-src alone (18 to 28 days). This cotransformation

ability was abrogated by deletions in CR1 or CR2 of E1A, which encode the binding regions for the pRb family

and are responsible for E1A-mediated cell cycle activation. Mutations in the p300 binding site or the second

exon, which abolish immortalization, did not affect v-src cooperation, in contrast to ras and adenovirus E1B.

While kinase activation was required for growth in soft agar, differential activation of Src kinase did not

correlate with transformation efficiency. Cell morphology and actin structures were not dramatically impacted

by E1A expression; thus, hypertransformation, as previously described for ras cotransformation, was not

observed with v-src and second-exon mutants of E1A. However, growth rates for cells expressing both E1A and

v-Src were higher than those for cells expressing only v-Src. These results suggest that functions involved in

cell cycle activation encoded by E1A first exon can enhance v-src transformation of primary epithelial cells.

Oncogenic transformation is a multistep process that

in-volves the loss of cellular growth controls maintained by tumor

suppressor genes, as well as the acquisition of enhanced

pro-liferative capacity and morphological alterations provided by

dominant oncogenes (for reviews, see references 4 and 85). In

support of this model, primary cells have been shown to be

resistant to transformation by dominant oncogenes acting

alone and seem to require the concerted action of at least two

oncogenes to become transformed (23, 46–48, 71; for a review,

see reference 37). Immortalized fibroblasts, on the other hand,

are much more sensitive than similar primary cells to

transfor-mation by a variety of dominant oncogenes, including v-ras and

v-src (21, 38, 55). The mechanism(s) behind this resistance to

transformation is beginning to be understood and may include

specific cell cycle blocks (30, 77, 82). In combination with the

fact that more than 80% of human solid tumors are of

epithe-lial origin, these observations suggest that primary epitheepithe-lial

cells would provide an excellent model system to study

molec-ular signals involved in carcinogenesis.

The adenovirus (Ad) E1A gene is one of several DNA

tu-mor virus oncogenes that have been used to study oncogene

cooperative transformation of primary cells (for a review, see

reference 2). E1A has been shown to immortalize primary

cells, such that they maintain many of their differentiated

char-acteristics (35, 64, 65). In addition, E1A can cooperate with

Ha-ras and Ad E1B, as well as polyomavirus mT (pmT), to

transform primary cells (14, 71, 81, 88). The pmT binds to and

activates the c-Src protein (5, 10). This c-Src stimulation is

required for cellular transformation by pmT (39). However,

pmT cannot transform primary cells without the assistance of

an immortalizing oncogene (11, 71). In the present study, we

have extended these observations by investigating the ability of

v-src to transform primary epithelial cells in the presence or

absence of E1A expression. When baby rat kidney (BRK) cells

were transfected with v-src, inefficient transformation was

ob-served. However, when v-src was cotransfected with Ad 5 E1A

12S, marked improvement in transformation efficiency was

noted. Synergistic enhancement of v-src transformation did not

require full immortalization functions of E1A but only

func-tions encoded by the conserved regions 1 and 2 (CR1 and

CR2), corresponding to the ability of E1A to activate quiescent

BRK cells into the cell cycle (65) and to the ability to bind the

pRb family of proteins (16, 89). Transformation by Ha-ras is

subject to dramatic modulation of transformation by E1A (13,

80). This was not observed with v-src cooperation.

v-src-trans-formed cells exhibited a loss of actin organization, in the

pres-ence or abspres-ence of E1A, and inhibition of v-Src with radicicol

(45) caused reestablishment of prominent actin stress fibers.

This suggests that the transformation by E1A and v-src may be

reversible and that the morphologic alterations observed were

due to the kinase activity of v-Src and not to functions encoded

by E1A. This is in contrast to transformation of primary

epi-thelial cells by Ha-ras and E1A. These data imply that

activa-tion of the cell cycle in primary epithelial cells by E1A is

sufficient to allow efficient oncogenic transformation of such

cells by v-src and that this transformation is not subject to

modulation by E1A.

MATERIALS AND METHODS

Cells, transfections, and plasmids. 293 cells are human kidney cells trans-formed by Ad E1A and E1B (26) and were maintained in Dulbecco modified Eagle medium (DMEM)–5% fetal calf serum (FCS). BRK cells were prepared from 5-day-old rats as previously described (71). Transfections on BRK cells were performed at 2 days postplating by a modification of the calcium phosphate precipitation technique (90), as previously described (71). pJH v-src (expressing the wild-type [WT] SH2 and SH3 domains fused to the COOH-terminal portion

* Corresponding author. Mailing address: Dept. of Microbiology

and Immunology, University of Tennessee Health Science Center,

Memphis, TN 38163. Phone: (901) 448-8219. Fax: (901) 448-8462.

E-mail: [email protected].

† Present address: Department of Cell Biology, The Scripps

Re-search Institute, La Jolla, CA 92037.

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of the Prague A v-src, which contains the kinase domain but lacks the regulatory tyrosine 527) was used at 5mg/60-mm-diameter dish, as were the E1A expression plasmids which have been described previously (13). To visualize transformed foci, cells were fixed with methanol at 14 days posttransfection and stained with Giemsa (Sigma, St. Louis, Mo.). For cell growth curves and Western analysis, whole transfected plates (.20 foci/plate) were trypsinized to establish a poly-clonal cell line and were used at low passage numbers (,10) in order to minimize the effects of clonal variation, except for v-src alone, which was derived from three separate clones that were pooled prior to assays. In addition, individual foci were cloned for additional growth curves and soft agar assays (Table 1). For cell growth curves, 105_{cells were seeded onto 60-mm-diameter dishes and grown in} DMEM–5% FCS for the indicated number of days. Each time point represents at least four cell counts with a hemacytometer. The curves represent two inde-pendent experiments. Radicicol was used as previously described (58, 93), except that incubation in radicicol was carried out for 12 h (for Western blots) or 24 h (for immunofluorescence).

For fluorescence-activated cell sorting (FACS) analysis, cells were maintained in normal growth medium for 2 days prior to analyses. A total of 106_{cells were} trypsinized, fixed with ethanol, and stained with propidium iodide for processing on a Coulter Profiler II analyzer (Coulter, Miami, Fla.). Phoenix Flow Multicycle analysis software was used to calculate the percentages of cells in G1, G2/M, and S phases in each population, as well as coefficients of variation (CV). The results represent at least two independent samples.

To test the ability of cell lines to grow independently of solid substrate, 60-mm-diameter dishes were coated with 2 ml of medium containing 0.5% agar (in DMEM-penicillin-streptomycin plus 5% FCS). The cells were then plated onto these dishes in the same medium at plating densities of 104_{, 10}5_{, and 10}6 cells per plate. The plates were incubated as described for cell line maintenance, with fresh medium (containing 0.5% agar) added every 3 to 4 days. Colony formation was assayed between 10 and 18 days after plating by overlaying the agar medium with 0.5 ml of p-iodonitrotetrazolium violet solution (0.5 g/liter in sterile water). Colonies were observed 24 h later. For samples exposed to radi-cicol, 0.1mg of radicicol per ml in agar medium was kept on cells, with fresh medium every other day.

Western blot analysis.Western blot analysis was performed essentially as described previously (58, 93). Briefly, approximately equal numbers of cells were labeled with (approximately 1mCi/23106_{cells) Trans-}35_{S-Label (ICN, Costa} Mesa, Calif.) overnight in DMEM with 5% FCS. The cells were washed twice with phosphate-buffered saline (PBS), lysed in boiling 23Laemmli sample buffer, scraped into tubes, and boiled for an additional 5 min. Lysates were centrifuged to remove debris, normalized to each other by scintillation counting, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon SP in a Bio-Rad Western blot apparatus. The filters were blocked overnight in blocking buffer (0.7% fish gelatin in 10 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20 [TBST]). The blots were probed with either anti-c-Src antibody (SC-019; Santa Cruz Biotechnologies, Inc., Santa Cruz, Calif.) or an antiphosphotyrosine antibody (Transduction Laboratories, Lexington, Ky.) in blocking buffer, followed by three washes with TBST. Anti-bodies were detected with anti-mouse-horseradish peroxidase conjugate (Sig-ma), followed by a chemiluminescence detection method, essentially as previ-ously described (58).

Immunofluorescence and photomicroscopy.Cells were seeded onto glass cov-erslips and maintained as described above. The cells were washed with cytoskel-eton buffer (32) and then fixed with 3.7% paraformaldehyde for 10 min at room

temperature, washed twice in cytoskeleton buffer, and permeabilized in 0.5% Triton X-100 in PBS. Nonspecific binding was blocked with 0.5% FCS–0.5% bovine serum albumin in PBS, and then cells were incubated in 1mg of phalloi-din-fluorescein isothiocyanate (FITC) per ml for 30 min, washed three times with PBS, and mounted in Airvol (Air Products, Allentown, Pa.). Fluorescence pho-tomicrographs were taken with a Zeiss Axiophot epifluorescence microscope fitted with an Optronics DEI-750 video camera system. For phase photomicros-copy, an Olympus CK2 microscope fitted with Minolta X-700 was used with Kodak Tri-X Pan ASA 400 film.

RESULTS

E1A first-exon functions enhance v-src transformation of

BRK cells.

To investigate possible cooperative transformation

of primary epithelial cells by E1A and v-src, a plasmid

express-ing v-Src was transfected alone or cotransfected with a plasmid

expressing either WT or mutated E1A 12S genes onto neonatal

BRK cells. Five functional regions of E1A have been

de-scribed, and mutations in all of these are represented by the

E1A mutants shown in Fig. 1 and have been previously

de-scribed (64, 88). As shown in Fig. 2, transformation of BRK

cells by v-src alone was inefficient; the visible foci that were

obtained were not observed until 18 to 28 days posttransfection

(data not shown). This is in contrast to Ha-ras transfections,

which never yield foci in the absence of E1A (71) (Fig. 2).

Transfections of v-src onto NIH 3T3 cells resulted in efficient

transformation (data not shown). When BRK cells were

co-transfected with v-src and WT 12S, efficient transformation was

observed, yielding numerous foci within 14 days

posttransfec-tion, similar to the transformation observed with Ha-ras (Fig.

2). All of the v-src-transformed cells tested had the ability to

grow as soft agar colonies, including the rare cells transformed

by v-src alone (Table 1). This cooperative transformation was

not abrogated by mutations in the second exon of E1A,

indi-cating that immortalization by E1A (which requires the second

exon) is not necessary for v-src transformation, in contrast to

E1A cooperation with Ad E1B (14, 15, 81). However,

muta-tions that have been shown to interfere with E1A’s ability to

stimulate cell cycle progression in quiescent cells also

inter-fered with v-src cotransformation. Specifically, deletion of the

NH2 terminus and CR1 (NTdl814, containing amino acids 83

to 243 [88]) or deletion of CR2 (dl891-1339, lacking amino

acids 110 to 174 [88]) of E1A abrogated cooperative

transfor-mation by v-src. Interestingly, pm563, which has been shown to

be unable to cooperate with Ha-ras (88), was still able to

cooperate with v-src (Fig. 2). pm563 does not form stable

complexes with the transcriptional coactivator p300 (16, 89).

However, this mutant does have the ability to stimulate cell

cycle progression, although the proliferation cannot be

main-tained (67). Thus, the ability of E1A to synergize with v-src to

transform primary epithelial cells is contained in the first exon

and correlates with activation of the cell cycle by E1A.

Expression and kinase activity of v-src is not consistently

altered by E1A.

E1A has been shown to affect expression of a

variety of cellular genes (for a review, see reference 2). To

investigate whether E1A had an effect on the overexpression of

v-src, cell lines were isolated from the transfections on BRK

cells and Western blot analysis was performed (Fig. 3). No

significant differences were observed in the expression of v-src

in the presence of WT or mutated 12S. Thus, the enhanced

transformation in the presence of E1A was not due to

differ-ential expression of v-src.

Some DNA tumor virus proteins have been demonstrated to

alter the activities of specific cellular kinases, such as

mitogen-activated protein (MAP) kinases (28) and c-Src (1). However,

the mechanisms for these effects are not clear. To test whether

the differential transformation efficiencies were due to changes

in v-Src activity, total cellular proteins were analyzed by

West-TABLE 1. Soft agar colony formation by transformed primary

epithelial cells

Cell line

Soft agar colony formation at the following plating densities:

104 ₁₀5 ₁₀6

src

2

2

1

src

1

WT 12S

1

1

1

src

1

HBdl12

1

1

1

src

1

CTdl976

1

1

1

ras

1

WT 12S

1

1

1

ras

1

HBdl12

1

1

1

WT 12S immortalized

2

2

2

src

1

RAD

₂

₂

₂

src

1

WT 12S

1

RAD

2

2

2

src

1

HBdl12

1

RAD

2

2

2

a_{Cells were inoculated into soft agar cultures as described in Materials and} Methods. Primary epithelial cells immortalized by WT 12S alone were included as a negative control. Colony formation was assayed by vital staining and mi-croscopy from two separate experiments.

b_{RAD, 0.1mg of radicicol per ml maintained in medium during the assay.}

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ern blotting (Fig. 4). The presence of v-Src resulted in a large

number of proteins being tyrosine phosphorylated, compared

to BRK cells expressing only WT 12S. While the total protein

phosphotyrosine content was higher in the WT

12S-plus-v-src-transformed cells, compared to the v-src-12S-plus-v-src-transformed cells, a

similar increase was not observed with CTdl976, which was as

competent as WT 12S in the transformation assays.

Inconsis-tent increases in c-Src tyrosine kinase activity in the presence

of DNA tumor virus oncogenes have been previously reported,

which also did not correlate with cellular transformation (1). In

the presence of the specific Src kinase inhibitor radicicol (45),

the levels of tyrosine phosphorylation were significantly

dimin-ished in all of the v-src-transformed cell lines tested. These

data suggest that v-Src is active in transformed BRK cells but

that levels of activity may not correlate with observed

trans-formation efficiencies. Kinase activity nonetheless seems to be

required for transformation, since incubation of or

v-src-plus-WT 12S-transformed BRK cells in the presence of

radi-cicol inhibits growth in soft agar (Table 1).

E1A enhances the proliferative properties of

v-src-trans-formed epithelial cells.

As shown above, of the five functional

regions of E1A, only the two regions required for cell cycle

activation are required to cooperate with v-src. Therefore, the

growth rates of BRK cells transformed with v-src or v-src plus

E1A (WT or mutant) were analyzed. As shown in Fig. 5A, cells

transformed by mutant or WT 12S and v-src had higher growth

rates and saturation densities, compared to primary epithelial

cells transformed by v-src alone. In normally cycling

popula-tions, the percentage of cells found in S phase was

approxi-mately 50% higher among BRK cells transformed by WT 12S

and v-src, compared to v-src alone (Fig. 5B). In the case of

dl891-1339 plus v-src, the resultant cells were not effectively

established into a cell line, such that they failed to proliferate

beyond early passages, which was exhibited in growth curves

(Fig. 5A). The dl891-1339-plus-v-src-transformed cells

ap-peared to experience a block in G2/M and/or G1

(Fig. 5B). This

was not a clonal aberrance, since the experiment was done with

a pool of multiple, independently arising foci. This may have

been due to loss of Src expression in these cells, since Src has

been shown to be required for G2

progression (70). It has been

previously shown that deletions of CR1 lead to a dominant

negative effect on the establishment of primary cells by WT

12S (50). In a similar manner, dl891-1339 may not allow the

stable establishment of primary cells, but this remains to be

determined. Thus, the ability to activate the cell cycle not only

is required for the initial transformation enhancement but also

has a significant effect on the growth properties of

v-src-ex-pressing cells.

E1A WT 12S is unable to modulate v-src transformation.

Functions encoded by the second exon of E1A have been

shown to down-modulate Ha-ras transformation of primary

epithelial cells, such that they retain some of their epithelial

morphology and characteristics (25). Deletion of the regions

encoding these functions leads to a marked increase in

trans-formation with ras (13, 80), but not E1B (14). This

hypertrans-formation is concomitant with dramatic alterations in cell

growth rates, adhesion, and actin cytoskeleton organization

(20a). To assess whether v-src transformation was subject to

such modulation imposed by E1A second-exon sequences, cell

morphologies and growth rates among cells transformed with

WT 12S plus v-src, CTdl976 plus v-src, and v-src alone were

compared. As shown in Fig. 6, none of the v-src transformants

maintained epithelial cell-style morphology or cell-cell

junc-tions; instead, they exhibited a star-shaped morphology, even

in the presence of WT 12S. Deletion of some or all of the

second exon did result in cells that were somewhat smaller and

more refractile and enabled them to grow to higher saturation

FIG. 1. Properties of E1A 12S mutants. The mutants have been described previously (64, 88) and are summarized here. Functional regions are indicated at the top. Protein binding indicates the ability of the protein to bind either Rb or p300 as well as WT E1A (1) or a level of binding that is less than that of WT (2). Data for the columns headed immort. (immortalized) and activation are derived from references 67 and 64; data for pRb and p300 are derived from references 89 and 24; data for ras were derived from references 88 and 13; data for E1B were derived from reference 74; and data for v-src were derived from the present study. aa, amino acids.

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densities (Fig. 5A), which is similar to what has been observed

in E1A cooperation with Ha-ras.

As shown in Fig. 5A, expression of E1A first-exon sequences

allowed cells to achieve much higher growth rates and

satura-tion densities than the cells transformed with v-src alone.

While deletion of the second exon did allow a modest

enhance-ment of saturation density over the WT 12S, the difference was

less than 50%. No differences were observed in the adhesive

properties of v-src-transformed cells, in the presence of either

WT or mutant 12S (data not shown). This is in contrast to

Ha-ras-transformed BRK cells, which are much more adhesive

in the presence of WT 12S expression, compared to

second-exon mutants of 12S (20a). These results indicate that

v-src-mediated cotransformation cannot be down-modulated in a

manner similar to Ha-ras cotransformation in primary

epithe-lial cells. The differences in the requisite functional regions of

E1A and in the potential for E1A to impact the extent of

transformation probably reflect the fundamental differences in

the signals generated by src, ras, and E1B.

v-src transformation of primary epithelial cells leads to a

loss of actin stress fibers, which are maintained in

E1A-im-mortalized BRK cells.

c-Src has been shown to be involved in

FIG. 2. Transformation of BRK cells by v-src and E1A mutants. Cells were transfected with plasmids expressing the various mutants of E1A and v-src, as described in Materials and Methods. The plates were fixed at 14 days posttrans-fection and stained with Giemsa and represent four or more dishes in at least two independent experiments.

FIG. 3. Analysis of v-src overexpression. Cells were cloned from the indi-cated transfections, and normalized lysates were processed for Western blotting and probed with an anti-Src antibody. 10_{BRK, primary BRK cells.}

FIG. 4. Expression of v-src leads to increased phosphorylation of cellular proteins and is inhibited by radicicol. Total cell lysates were prepared from the cells indicated, which had been incubated in the presence (1[lanes 5 to 7]) or absence (2[lanes 1 to 4 and 8 to 10]) of radicicol (rad; 0.1mg/ml) for 12 h prior to lysate preparation. The blot was probed with antiphosphotyrosine antibody. Lanes 8 to 10, lower exposure of lanes 2 to 4.

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the assembly of focal adhesion complexes and activation of

integrin-mediated signals, primarily due to its ability to form

stable complexes with FAK (40, 74, 75). c-Src kinase activity is

involved in the regulation of epidermal growth factor

(EGF)-dependent, actin cytoskeleton rearrangement via

phosphoryla-tion of rhoGAP p190 (7). v-Src, on the other hand, has been

demonstrated to cause disassembly of actin stress fibers and

focal adhesions in fibroblasts (20, 42). Similarly, other viral

oncoproteins have been observed to affect actin stress fibers

(59, 86). E1A has been shown to induce and maintain

epithe-lial cell differentiation and morphology (Fig. 7C) (8, 22, 25, 64,

66). Therefore, the organization of F actin in

src-plus-E1A-transformed versus E1A-immortalized BRK cells was

ana-lyzed.

Actin stress fiber organization was observed in BRK cells

transformed with v-src and E1A via phalloidin-FITC staining,

followed by immunofluorescence. As shown in Fig. 7, cells

expressing v-src exhibited a significant loss of actin stress fibers,

compared to either the primary BRK cells or

E1A-immortal-ized cell lines, as well as increased amounts of actin

micro-spikes at the cell periphery. In contrast, both the primary cells

and E1A-immortalized epithelial cells maintained prominent

actin stress fibers, as well as anti-paxillin-staining focal

adhe-sion plaques, which were also lost in v-src-transformed cells

(data not shown). Actin microspikes were not observed in

primary or immortalized epithelial cells. When

E1A-plus-v-src-transformed BRK cells were exposed to radicicol prior to

phal-loidin staining, prominent actin stress fibers were again

ob-served, although complete epithelial morphology was not

regained. In addition, while all v-src-expressing cells grew in

soft agar assays, cells maintained in radicicol did not form soft

agar colonies (Table 1). Thus, the cytoskeletal alterations

ob-served in v-src-plus-E1A-transformed BRK cells correlate with

substrate independence. Furthermore, these alterations in

ac-tin organization are due entirely to v-Src kinase actions, not to

functions provided by E1A. This differs from E1A plus Ha-ras

transformation of BRK cells, in which case the morphology

and actin cytoskeleton of the resulting transformed cells can be

affected by specific mutations in E1A (13, 20a, 25). In the case

of Ad E1B-transformed epithelial cells, the actin cytoskeleton

is not disrupted in the presence of WT or mutated E1A (data

not shown). Thus, alterations of the actin cytoskeleton of

v-src-transformed primary epithelial cells are not subject to

modu-FIG. 5. (A) Cell growth curves. Cells from the indicated transfections were seeded at 105_{cells per dish, and growth rates were measured as described in Materials} and Methods. Each data point represents an average of four counts. (B) FACS analysis of cells transformed with v-src, v-src plus dl891-1339, and WT 12S plus v-src. Cells were stained with propidium iodide and sorted as described in Materials and Methods.

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FIG. 6. Cell morphologies of transformed or immortalized (imm) BRK cells. Photomicrographs were taken at equal magnifications. Bar, 100mm; 10_{BRK, primary} BRK cells.

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lation by WT 12S, but direct inhibitors of Src kinase can

mod-ulate such actin reorganization.

DISCUSSION

The results presented here show that transformation of

mammalian primary epithelial cells by v-src alone is inefficient,

in contrast to primary fibroblasts (34) or established fibroblast

lines such as NIH 3T3 cells. However, coexpression of E1A

leads to efficient transformation of these cells, such that they

are able to grow indefinitely and in an anchorage-independent

fashion. This synergistic cotransformation ability mapped to

the sequences contained in the first exon, in particular, CR1

and CR2 of E1A 12S. Interestingly, a deletion mutant lacking

CR2 (dl891-1339) produced few transformed foci, which did

not produce stable cell lines. Furthermore, an NH2-terminal

deletion mutant lacking CR1 (NTdl814) did not produce

formed foci with v-src. That the latter did not enable src

trans-formation was not surprising, since our lab has previously

found NTdl814 to actually inhibit WT 12S functions (50).

These observations underscore the requirement for CR1 and

CR2 functions for v-src transformation. An NH2-terminal

point mutant, pm563, which lacks the ability to bind p300 (89),

was able to enhance v-src transformation, indicating that the

requirements for v-src cooperation are less stringent than those

for E1B or Ha-ras cotransformation (14, 79, 88). Indeed,

nei-ther E1B, Ha-ras, nor pmT, which activates c-src (5, 10, 39), is

able to transform primary cells alone, but each requires

mul-tiple but different functions encoded by E1A (14, 71, 79, 81,

88). Nevertheless, v-src alone is able to transform primary

epithelial kidney cells, albeit inefficiently. Previous studies have

shown that primary rodent (but not human) fibroblasts can be

transformed by v-src alone (34). Susceptibility to

transforma-tion by v-src in primary cells appears to be partly dependent on

cellular senescence and aging (82). Also, it has been shown

that adrenocortical cells can be inefficiently transformed by

v-src and that v-myc can cooperate with v-src to efficiently

transform such cells (48). The inefficient transformation by

v-src alone in this study as well as previous studies could rely on

the acquisition of mutations in cell cycle regulatory genes

po-tentially reproducing the effects of E1A expression. While this

hypothesis remains unconfirmed, heterogeneity in primary

cul-tures may be relevant to the observation of rare v-src

transfor-mants (83). It seems likely that the inefficiency of

transforma-tion observed here may be due at least in part to the fact that

BRK epithelial cells undergo senescence and apoptosis after

24 h in culture, which E1A is able to rescue (63). In addition,

the ability of E1A, via CR1- and CR2-mediated binding of the

pRB family (6, 89), to activate the cell cycle probably enables

FIG. 7. F-actin organization of E1A-immortalized epithelial cells versus E1A-plus-v-src-transformed epithelial cells. Cells were stained with phalloidin-FITC, as described in Materials and Methods. (A and B) WT 12S-plus-v-src-transformed cells; (C) WT 12S-immortalized cells; (D and E) HBdl12-plus-v-src-transformed cells; (F) HBdl12-immortalized cells; (G and H) v-src-transformed cells; (I) primary BRK cells; (B, E, and H) cells exposed to 0.1mg of radicicol per ml for 24 h prior to fixation. Bar, 30mm.

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the somewhat higher growth rates observed in the E1A- and

src-transformed cells, compared with the cells transformed by

src alone. The ability of E1A to activate quiescent cells into the

cell cycle seems crucial for the enhancement of v-src

transfor-mation of primary epithelial cells.

Hyperphosphorylation of cellular proteins by v-Src seems to

be enhanced by WT 12S, but not by mutants lacking the second

exon, such as CTdl976. The significance of this is not clear at

present, since CTdl976 is as competent in transformation

as-says with v-src as WT 12S. Furthermore, the enhanced kinase

activity does not correlate with substrate-independent growth

or growth rate in vitro (Fig. 5; Table 1). Other investigators

have observed inconsistent increases in tyrosine

phosphoryla-tion by src in cells expressing E1A (1), but the data neither

indicate the significance of nor suggest a mechanism for the

increase. We find here that the mechanism does not involve

increased expression of the v-src oncogene. However,

differ-ences in localization, stability, or specific activity have not been

established at present.

Requirements for transformation of primary epithelial cells

by v-src and E1A are different from those of other cooperating

oncogenes (Fig. 1). For efficient transformation of primary

epithelial cells by v-src and E1A, the ability of E1A to induce

cell cycle via CR1 and CR2 is apparently the only requirement.

This is interesting, because previous studies have suggested

that immortalization of primary cells might be required for

efficient v-src transformation (38, 48), while we have observed

here that mutants incapable of immortalization by themselves

are able to cooperate with v-src to transform primary epithelial

cells (Fig. 1). Thus, E1A mutants lacking the ability to establish

cell lines alone can cooperate with v-src to establish

trans-formed lines. In fact, the requirements for E1A to cooperate

with v-src in transforming primary epithelial cells are less

strin-gent than those for Ad E1B or even Ha-ras. This is further

reflected in the ability of v-src to transform such primary cells

alone, albeit inefficiently, while neither Ad E1B nor Ha-ras is

able to do so (47, 71).

The role of p300 binding by E1A in cell transformation has

been found to involve changes in gene expression (for reviews,

see references 2 and 54). Similarly, changes in cellular gene

expression can also be induced by v-src transformation (27, 52,

76). Furthermore, it has been shown that p300 binding by E1A

can contribute to cell proliferation (36, 92), by interfering with

the expression of p21

_{(3, 12, 53, 87). p27}

_{and p21}

are cyclin-dependent kinase inhibitors with many targets in

common (9, 17, 19, 31, 41, 56, 60, 61, 84). However, p27

_can

be inhibited by E1A directly, apparently independently of

tran-scription alterations (49). It is possible that primary kidney

epithelial cells (as opposed to the PC12 cells used in the

afore-mentioned studies) are prevented from proliferation primarily

by p27

_{and not p21}

_{, although this has not been formally}

tested. Since p300 binding does not seem to be required for

E1A’s cooperative transformation with v-src but is required for

ras cotransformation (for a review, see reference 2), it seems

possible that v-src may allow cells to escape a proliferation

block by a p21

_{-dependent mechanism. Given that p27}

and p21

_{have been shown to be involved in}

attachment-dependent growth (19) and that inhibition of Src kinase activity

prevents soft agar growth (Table 1), it is possible that Src

kinase activity allows cells to circumvent such cell cycle

regu-lation steps, as has been suggested elsewhere (72).

The data presented here also show that while E1A is able to

enhance transformation efficiency, v-src-mediated

morpholog-ical alterations are not affected dramatmorpholog-ically by E1A. These

alterations include loss of cell-cell contact, actin stress fibers

and/or microfilaments, and focal adhesion plaques. Radicicol,

a potent and reversible inhibitor of Src kinase activity (44, 45,

58), is able to reverse the loss of actin stress fibers induced by

v-Src, although cells do not regain cell-cell contacts. Although

radicicol has been shown to be specific to Src kinase (45), it can

inhibit mos and ras transformation as well (93). The fact that

v-src-transformed primary epithelial cells exhibit loss of stress

fibers is hardly surprising, given the wealth of data showing this

phenomenon in other cell types (for a review, see reference 57

and references therein). However, c-Src has a positive effect on

cell spreading and focal adhesion formation on fibronectin, but

this role of c-Src does not seem to require its kinase activity

(40). However, it is interesting that E1A is not able to suppress

v-src-mediated morphological transformation of epithelial

cells, given E1A’s ability to do so with an activated ras gene

(13, 20a, 25).

The ability of v-src alone (present study and reference 34)

but not Ha-ras (present study and references 47 and 71) to

transform primary cells suggests further differences between

the signals mediated by ras and src. Both ras and src activation

cause stimulation of the ERK-MAP kinase cascade via c-raf

activation (18, 51, 91). The mechanisms employed by Src and

Ras to accomplish this seem to be different (78). The activation

of raf, however, does not lead to full transformation of either

NIH 3T3 cells (43, 62) or primary epithelial cells, even with

E1A (20a), and activation of rho-related proteins is required in

combination with the activation of raf for full transformation.

The activation of Rho family proteins by ras is well

docu-mented (for a review, see reference 68). src is also able to

transduce signals through the Rho family G proteins via their

GAPs (7, 33). However, activated ras and activated src have

been shown to have opposite effects on actin stress fibers in

fibroblast cells, presumably by their actions on Rho activity (7,

69). In BRK cells, however, cotransformation by either Ha-ras

or v-src leads to a loss of stress fibers, again suggesting common

cellular effects for these oncogenes, although the mechanisms

may be different. Perhaps the differences in their abilities to

transform primary epithelial cells alone, or in conjunction with

E1A, can be explained by the observation that src can act

upstream of ras, as some signal transduction cascade models

would suggest (73). This might indicate that the activities or

signals induced by activated ras are but a subset of those

induced by activated src in epithelial cells. In addition, since

Src mediates important alterations in epithelial cell

transfor-mation directly by its kinase activity (29), it may be a more

potent oncogene than ras in epithelium-derived cells (48).

ACKNOWLEDGMENTS

We thank A. Reynolds for the gift of the pJH v-src plasmid and S.

Sharma for the radicicol. The technical expertise provided by M.

Dock-ter and J. Hermann and graphics assistance of T. Higgins of the UT

Molecular Resource Center are also appreciated.

This work was supported by the National Institutes of Health, and

R.S.F. was supported by the Regan Memorial Fellowship.

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